Recombinant Rhizobium bacteria inoculants

ABSTRACT

The sequences of a Rhizobium bacteria responsible for competitiveness with respect to plant nodulation have been isolated and permanently transferred to superior nodulating Rhizobium genome. This has resulted in a stable construct that can form a plant inoculant that yields effective nodulation, while reducing the risk of suppression by other bacteria in the environment.

This invention was made with U.S. government support awarded by the U.S.Dept. of Agriculture (USDA), Grant #(s): 89-37262-4792, 87-CRCR-1-2571and HATCH Funds. The U.S. Government has certain rights in thisinvention.

This application is a continuation of application Ser. No. 07/358,744,filed May 30, 1989, now abandoned.

BACKGROUND OF THE INVENTION

1. Field Of The Invention

The present invention relates to recombinant DNA technology. It appearsespecially useful for improving the nodulation (and thus nitrogenfixation) capability of plants.

2. Description Of The Art

Root nodule Rhizobium bacteria are responsible for symbiotic nitrogenfixation in the nodules of certain plants (e.g. legumes). Where naturalbacterial activity is ineffective, the plants must rely on the existingnitrogen in the soil or on fertilizers. Where the former occurs, thequality of the soil is reduced. Where the latter occurs, the cost to thefarmer (and ultimately the public) can be substantial. Further, the useof fertilizers often raises environmental concerns.

It is now known that the presence of certain "inferior" strains ofRhizobium in soil can depress the productivity of not only other naturalbacteria, but also of "superior" bacteria added by inoculation of seeds.This can frustrate attempts to inoculate seeds prior to planting or toinoculate roots during plant growth. When inoculation has beensuccessful, it is usually because the indigenous bacterial populationshave been small.

Many investigators have studied the factors involved in determiningnodule occupancy by strains of Rhizobium. See e.g. D. Dowling et al., 40Annu. Rev. Microbiol. 131-157 (1986) (the disclosure of this article andof all other articles referred to herein are incorporated by referenceas if fully set forth). Despite this work, no solutions to the abovedescribed Rhizobium competition problem have been developed.

In E. Triplett et al., 85 Plant Physiology 335-342 (1987) and 11th NorthAmerican Rhizobium Conference Abstract GP4 (1987), my laboratoryreported on the fact that the Rhizobium leguminosarum bv. trifoliibacterial strain T24 appeared to have genes in its coding responsiblefor a suppressor of other Rhizobium (I named the substance trifolitoxin)and other genes coding for T24's own resistance to trifolitoxin'seffects. Unfortunately, I also have found that trifolitoxin productionby transconjugant bacterial cells that I had constructed was readilylost in the absence of tetracycline. Thus, the earlier Rhizobiumtransconjugants were not likely to be able to effectively limitnodulation by trifolitoxin-sensitive indigenous strains of Rhizobiumunder agricultural conditions (where tetracycline application isimpractical).

It was therefore desired to more specifically isolate and characterizethe genes responsible for the T24 suppressor and resistancecharacteristics and use information developed therefrom to find a meansfor stably inserting such genes in the genome of "superior" Rhizobium soas to ultimately lead to a Rhizobium that can form effective nodulesnotwithstanding the presence of indigenous strains.

SUMMARY OF THE INVENTION

The invention provides a recombinant Rhizobium bacteria capable ofassisting in the formation of nitrogen fixation nodules on at least someplants. The bacteria has a foreign sequence expressibly coding fortrifolitoxin. The bacteria preferably also has a sequence coding forresistance to trifolitoxin suppression, with both foreign sequencesbeing in the bacterial genome. The term "genome" is used herein to referto either the bacterial chromosome or other bacterial genetic sequencesin the bacteria.

Inoculants can be provided that use these bacteria. Thus, plant seeds(or the roots of young plants) can be inoculated with the bacteria.

Further, plant cells can be formed that incorporate these sequences (sothat the plant strain produces its own trifolitoxin). In thealternative, a production host can produce trifolitoxin on a commercialscale. In either case, the trifolitoxin can be used as a trans inoculantby having the superior strain have the resistance gene only.

It will be appreciated that the invention provide the ability toeffectively create nitrogen fixation nodules in the presence of inferiorstrains.

The objects of the invention therefore include:

A. providing a recombinant bacteria of the above kind;

B. providing a recombinant host of the above kind;

C. providing a plant seed inoculated with a bacteria of the above kind;and

D. providing a plant inoculant using a bacteria of the above kind.

These and still other objects and the advantages of the presentinvention will be apparent from the description that follows.

DESCRIPTION OF THE PREFERRED EMBODIMENTS General Overview

Rhizobium leguminosarum bv. trifolii T24 induces ineffective nodules butproduces a potent anti-rhizobial compound, trifolitoxin. As a result oftrifolitoxin production, T24 prevents root nodulation bytrifolitoxin-sensitive bacterial strains. The main objective of thiswork was to identify and isolate the trifolitoxin production andresistance genes and permanently transfer those gene to other strains ofRhizobium that produced "superior" nodules.

To achieve this, a genomic library of T24 was prepared in the prior artcosmid vector pLAFR3. One cosmid clone was identified that restoredtrifolitoxin production and nodulation competitiveness. We formed arecombinant plasmid from this cosmid clone, pTFX1, that conferredtrifolitoxin production and resistance on other bacteria (albeit in anunstable fashion).

Transposon mutagenesis and restriction analysis was then used to map andsubclone the insert of pTFX1. A 4.4 kb region of DNA, referred to as tfxwas found to be necessary for the expression of trifolitoxin productionand resistance in Rhizobium. Another portion was found to havesufficient homology to Rhizobium genome to permit the use of a techniquefor insertion into the genome. Several mutants of pTFX1 (with Tn5insertions outside the trifolitoxin region) were therefore used topermanently insert the trifolitoxin genes into several strains ofRhizobium. This resulted in a stable construct having the desiredcharacteristics.

METHODS AND MATERIALS

The identification of the precursor cosmid clone, and the formation ofplasmid pTFX1 is described in detail in my article, E. Triplett, 85P.N.A.S. USA 3810-3814 (June, 1988) (not prior art). I then made arestriction map of pTFX1. I did this by restriction analysis of Tn5insertions in pFFX1. This map was used to determine the size andlocation of the trifolitoxin genes as well as to develop a strategy tosubclone the trifolitoxin genes into the broad host range vector,pRK415, N. Keen et al., 70 Gene 191-197 (1988). I found that the abilityof pTFX1 to confer trifolitoxin production as well as resistance intrifolitoxin-sensitive strains of Rhizobium were located within a 4.4 kbregion of pTFX1, this knowledge, plus my analysis of the other portionsof the insert in turn led to selection of the marker exchange techniquefor inserting these genes in a bacterial genome.

In my work, Rhizobium strains were cultured at 28° C. on Bergersen'ssynthetic medium (BSM) as described by F. Bergersen, 14 Aust. J. Biol.Sci. 349-360 (1961). Strains of E. coli were cultured at 37° C. onLuria-Bertani (LB) medium. Antibiotics were added as needed at thefollowing final concentrations: kanamycin (Km), 50 ug/ml; tetracycline(Tc), 12.5 ug/ml; spectinomycin (Sp), 50 ug/ml; streptomycin (Sm), 50ug/ml; gentamycin (Gm), 25 ug/ml; nalidixic acid (Nal), 10 ug/ml; andneomycin (Nm), 75 ug/ml.

Conjugation of the plasmid mutants (e.g. pTFX1::Tn5) into Rhizobium wasperformed using procedures analogous to those described in E. Triplettet al., 85 Plant Physiol. 335-342 (1987) with some modifications. Inthis regard, the donor, recipient, and helper strains were mixed in a1:1:1 ratio in water each at a cell density of approximately 5×10⁷ perml. After vortexing, a 5 ul suspension of this mixture is placed on aYM/KB (see E. Triplett (1987), supra) plate with 3% agar. Afterincubation for two days at 28° C., each mating was resuspended in 0.1 mlwater and spread plate on a BSM plate prepared with noble agar andsupplemented with tetracycline and streptomycin. The use of noble agarin the interruption media eliminated the background of growth on theplates. After five days, transconjugants were observed.

Conjugations involving the transfer of plasmid DNA between strains of E.coli were done as described above except that 5 ul of the mixture ofdonor, recipient, and helper strains were placed on an LB plate andincubated at 37° C. overnight. Interruptions were done as describedabove with the appropriate selective media on solid LB medium.

In the transfer of plasmid DNA from E. coli to Rhizobium, E. coli DH5a(Bethesda Research Labs) (pRK2013), D. Figurski et al., 76 P.N.A.S. USA1648-1652 (1979), was used as the helper strain. In the transfer ofplasmid DNA between two strains of E. coli, E. coli HB101, H. Boyer etal., 41 J. Mol. Biol. 459-472 (1969), (pRK2073) (S. Leong et al., 257 J.Biol. Chem. 8724-8730 (1982)) served as the helper strain.

Large scale plasmid preparations were purified by the boiling methoddescribed by D. Holmes et al., 114 Anal. Biochem. 193-197 (1981). Forrestriction analysis of small amounts of plasmid DNA, plasmids werepurified from cells grown on sold medium by the alkaline lysis miniprepmethod described by F. Ausubel et al., Current Protecols In MolecularBiology (1987).

The recombinant plasmid, pTFX1, was mutagenized with Tn5 by the methodof G. Ditta, 118 Meth. Enzmol. 519-528 (1986) with slight modifications.The plasmid pTFX1 was transformed into E. coli cell line HB101::Tn5 asdescribed by D. Hanahan, 166 J. Mol. Biol. 557-580 (1983) using LBmedium supplemented with kanamycin and tetracycline for selection oftransformants. The transformants were pooled and conjugated with HB101(pRK2073) and C2110nal (Ditta, supra). The triparental matings wereincubated overnight at 37° C. Cells were resuspended in water and adilution series plated on LB medium supplemented with kanamycin,tetracycline, and nalidixic acid. Transconjugants were pooled andplasmid DNA isolated by an alkaline lysis miniprep procedure asdescribed by F. Ausubel et al., Current Protocols In Molecular Biology,John Wiley & Sons, New York (1987). Fourteen separate matings wereperformed in order to enhance the prospects of obtaining independentmutations. Plasmid DNA was transformed into E. coli DH5a and thesubsequent transformants selected on LB medium with kanamycin andtetracycline.

RESTRICTION ANALYSIS

Restriction analysis of three hundred and thirty-six pTFX1::Tn5 mutantswas done to provide the information necessary to construct a restrictionmap of pTFX1. Each mutant was also conjugated into R. leguminosarum bv.trifolii strain TA1 as described above to determine the trifolitoxinphenotype. (The tfx genes are not expressed in E. coli.)

Plasmid DNA of each pTFX1::Tn5 mutant was cleaved with the followingrestriction enzymes: Eco RI, Kpn I, Dra I, and Mlu I. To accurately mapTn5 insertions within each restriction fragment, selected pTFX1::Tn5plasmids were cleaved with Hpa I, an enzyme with two symmetricalrestriction sites within the inverted repeat sequence elements of Tn5.Hpa I has two restriction sites in pTFX1. This enzyme was used for thispurpose rather than Bgl II since there are no Bgl II restriction sitesin pTFX1. Plasmid DNA was electrophoresed in 0.6% agarose at 100 v. Forthe separation of fragment sizes greater than 15 kb, field inversionelectophoresis was used. At 0.3 s intervals, the electric field wasinverted between 100 v toward the anode and 60 v toward the cathode.Field inversion gels were run for 16 hours at 4° C. All gels were 10 cmin length.

The ability of a strain to produce trifolitoxin was determined bybioassay; Southern analysis was determined with biotinylated probes ofeither pLAFR3 or pTFX1; and trifolitoxin was partially purified from thecell culture supernatants of T24 and various Rhizobium transconjugantscontaining pTFX2 by reverse phase chromatography; these steps all beingdone as described in the general technique portion of E. riplett, 85P.N.A.S. USA 3810-3814 (June, 1988) (not prior art).

For example, a bioassay for trifolitoxin appearing at page 3811 of thatarticle is as follows:

A suspension of a trifolitoxin-sensitive strain of R. leguminosarum bv.trifolii in water was diluted to an OD of 0.1 at 600 nm, and 0.1 ml wasspread on 100 mm (maximum diameter) plates containing BSM agar. Asterile cork borer was used to cut a 8 mm diameter hole in the center ofthe agar. In this hole was placed 100 μl of filter-sterilized culturefluid supernatant or a partially purified sample of trifolitoxin.

When screening transconjugants from the genomic library for trifolitoxinproduction, a suspension of each transconjugant was prepared in water; 5μl of each suspension was placed on a BSM plate containing tetracyclineat 12.5 μg/ml. After 24 hours at 28° C., each plate was sprayed with asuspension of a tetracycline resistant derivative of R. leguminosarumbv. trifolii 2046, which was prepared by conjugating pRK415, a plasmidthat confers tetracycline resistance, into 2046. The conjugation wasperformed with pRK2013 as the helper plasmid. After 36 hours at 28° C.,zones of inhibition were observed in strains carrying plasmids thatconferred trifolitoxin production.

When screening recombinant or wild-type strains for trifolitoxinproduction, suspensions of test strains were prepared and diluted to anOD of 0.1 at 600 nm. These were spread (0.1 ml) on BSM plates andallowed to dry for one hour. Suspensions of each trifolitoxin-producingstrain were prepared in water and diluted to an OD of 0.5 at 600 nm.Five microliters of each suspension was placed in the center of a driedBSM plate. After 48 hours at 28° C., zones of inhibition were measured.

From this analysis, I determined that my previous estimate of the sizeof the insert in pTFX1, 24.2 kb was inaccurate. The insert size in pTFX1is now known to be 29.5 kb.

The restriction analysis showed that two enzymes, Dra I and Mlu I, didnot have restriction sites in either Tn5 or tfx. The tfx region resideson a 10 kb Dra I fragment and a 7.5 kb Mlu I fragment. Since tfx ispresent on a smaller fragment in the Mlu I digest than in the Dra Idigest, Mlu I fragments were chosen for subcloning tfx.

SUBCLONING

One mutant of pTFX1 was chosen whose Tn5 insertion was located withinthe 7.5 kb Mlu I fragment of pTFX1, yet did not affect the expression oftrifolitoxin production in Rhizobium. Ligation of the Mlu I fragmentsfrom which contains both the intact trifolitoxin production genes and aTn5 insertion on the same fragment, to the broad host-range vector,pRK415, allows for selection against the other possible ligationproducts.

An Mlu I digest of plasmid DNA from a pTFX1::Tn5 mutant was blunted withT4 DNA polymerase using techniques described by F. Ausubel et al.,Current Protocols In Molecular Biology, John Wiley & Sons, New York(1987). These fragments were ligated to an alkaline phosphatase-treatedXmn I digest of pRK415. The resulting ligated DNA was transformed intoDH5a competent cells and transformants selected on LB solid mediumsupplemented with kanamycin and tetracycline. Restriction analysis ofthe plasmid DNA of a selected transformant showed an insert size of 13.2kb (as was predicted based on the size of an Mlu I fragment of pTFX1with a Tn5 insertion). This plasmid is referred to as pTFX2.

A restriction map of pTFX2 was prepared based on the restriction sitesknown to be present in pRK415, the Eco RI restriction sites present inthe Mlu I fragment in pTFX1, and on double restriction digests of pTFX2with Sst I, Eco RI, and Hpa I. The Xmn I site in pRK415 and the Mlu Isites in the insert were eliminated by the blunt end ligation of theinsert into the vector.

To determine whether pTFX2 possessed functional tfx, this plasmid wasconjugated into Rhizobium. Trifolitoxin production was observed by theresulting transconjugants and confirmed using techniques describedpreviously for pTFX1 transconjugants in E. Triplett 1988, sucra (notprior art).

INSERTION INTO BACTERIAL GENOME

As an example of inserting tfx into a selected bacterial genome, themethod of G. Ditta, 118 Meth. Enzmol. 519-528 (1986) was adapted for R.leguminosarum bv. trifolii TA1. (A. Gibson, CSIRO) The technique startsfrom the idea that certain plasmids may be incompatible with certainother plasmids in certain hosts, and that under antibiotic stress thehost will tend to either drive one out (or hopefully where homologyexists take in the unwanted genetic material as part of the bacterialgenome). The incompatible plasmid pPH1JI (J. Beringer, 276 Nature633-634 (1978)) was conjugated into several TA1 transconjugants with mypTFX1::Tn5. It will be appreciated that the host Rhizobium can be other"superior" hosts of interest. The conjugation was interrupted on BSMprepared in noble agar and supplemented gentamycin, kanamycin, andspectinomycin. The resulting exconjugants (with the gene in the cellgenome) were replica-plated on BSM with tetracycline. Thetetracycline-resistant strains were discarded.

Bacterial strains T24, TA1 (pTFX1), and trifolitoxin-producing TA1(pTFX1::Tn5) transconjugants and TA1::TFX::Tn5 exconjugants werestreaked to single colonies on BSM medium in the absence of selectiveantibiotics. After two days of incubation at 28° C., a portion of theconfluent growth on the plate was suspended in water and 5 ul of thatsuspension spotted in the center of a BSM plate for the assay oftrifolitoxin production. A single colony from the initial plate was usedto inoculate a second plate. After two days, confluent growth on thesecond plate was used to assay trifolitoxin. The assays continued for 10"generations" or until trifolitoxin production was no longer observed.TA1::TFX:Tn5 showed stability through ten generations.

It will be appreciated that the present invention involves, inter alia,the location of the trifolitoxin production and resistant genes, thecloning of them, and the development of a way to insert them permanentlyin the bacterial genome.

Cultures of pTFX1::Tn5 (a/k/a pTFX1:10-15) in E. coli and RhizobiumTA1::TFX:Tn5 (a/k/a TA1::10-15) are on deposit at the American TypeCulture Collection, Rockville, Md., U.S.A., with ATCC numbers 67990 and53912 respectively. They will be made available upon issuance of thispatent and as provided under U.S. and other applicable patent laws.However, this availability is not to be construed as a license to usethe invention.

The preferred way to use the preferred bacteria is to streak thedeposited TA1::10-15 on BSM solid AGAR and wait for 2-3 days. One thenstreaks the growth product into BSM liquid broth. After several moredays one can pour the liquid broth on peat and uses the peat as acarrier to surround the seeds or roots. Note also that other knowncommercial inoculant techniques can readily be adapted for use withthese bacteria. See e.g. R. Roughley et al. in Nitrogen Fixation InLegumes, p 193-209 (1982); resulting in inoculants and inoculated seeds.This invention appears most likely to be useful on clover, peas, beans,vetch, and soybeans, but may well have utility wherever Rhizobiumcreated nodules.

Another possible use of the invention is to insert only the resistancegene in a bacteria and then add trifolitoxin to the soil (or transform aplant cell so it produces the trifolitoxin). In this regard, severalvectors are already known that can expressibly transform a plant genome,and many commercial production hosts are known.

It will be appreciated that various other changes to the preferredembodiment may be made. For example, various other strains besides T24may produce trifolitoxin, and thus their sequences could be used (e.g.after location with a hybridization probe based on pTFX1). Also, meansof inoculating the roots of live plants (as opposed to just seeds)during transplantation can easily be developed using known techniques.Further, other means for inserting the foreign genes in the bacterialchromosome may prove useful. See e.g. G. Barry, 4 Bio/Technology 446-449(1986) and 71 Gene 75-84 (1988). The claims should therefore be lookedto to judge the full scope of the invention and the preferred embodimentis not to be considered as representing the full scope of the invention.

I claim:
 1. A recombinant Rhizobium bacterium that is capable ofassisting in the formation of nitrogen fixation nodules on at least someplants, the bacterium having a foreign DNA sequence inserted in itsnatural bacterial genome expressively coding for trifolitoxinproduction, the foreign DNA sequence also coding for resistance totrifolitoxin, said trifolitoxin being a rhizobial proteinaceous materialthat is encoded for by a nucleotide sequence present in a 4.4 kb DNAfragment of R. leguminosarum bv. trifolii T24 that is in a pTFX1 portionof ATCC 67990, and said DNA sequence coding for resistance totrifolitoxin having a nucleotide sequence that is present in said 4.4 kbDNA fragment of R. leguminosarum bv. trifolii T24 that is in a pTFX1portion of ATCC 67990, wherein the bacterium is selected from the groupconsisting of Rhizobium leguminosarum, Rhizobium fredii, and Rhizobiummeliloti.
 2. A recombinant Rhizobium bacterium that is capable ofassisting in the formation of nitrogen fixation nodules on at least someplants, the bacterium having a foreign DNA sequence inserted in itsnatural bacterial genome coding for resistance to trifolitoxin, saidtrifolitoxin being a rhizobial proteinaceous material that is encodedfor by a nucleotide sequence present in a 4.4 kb DNA fragment of R.leguminosarum bv. trifolii T24 that is in a pTFX1 portion of ATCC 67990,and said DNA sequence coding for resistance to trifolitoxin having anucleotide sequence that is present in said 4.4 kb DNA fragment of R.leguminosarium bv. trifolii T24 that is in a pTFX1 portion of ATCC67990, l wherein the bacterium is selected from the group consisting ofRhizobium leguminosarum, Rhizobium fredii, and Rhizobium meliloti.